Abstract Body:
Introduction: Immune thrombocytopenia (ITP) is an autoimmune disorder wherein platelets are targeted by the patient’s own immune system, leading to low platelet counts, increased bleeding risk and compromised vascular integrity. Besides antibodies, cytotoxic CD8 T cells can contribute to the autoimmune attack involving the MHC-I complex. (TREM)-like transcript 1 (TLT-1), a platelet expressed protein, plays a role in platelet activation and sepsis. We previously discovered the interaction of TLT-1 with CD3e on T cells in cancer. However, the mechanistic role of TLT-1 in immune thrombocytopenia is not understood.
Hypothesis: TLT-1 suppresses MHC-I dependent activation of T cells and protects against T cell mediated platelet dysfunction in ITP.
Methods: Human blood samples (n=15) were used for ex-vivo assays while in vivo studies utilized a mouse model of ITP. T cell studies were performed using flow cytometry, ELISpot, and transcriptomics to determine the effect of TLT-1. Computational modeling, molecular docking and in-silico data analysis were used to study TLT-1 interactions with CD3.
Results: Platelet dysfunction (severe apoptosis) and CD8 T cell hyperactivation were demonstrated in ITP patients. This is clinically relevant as ITP patients with poor prognosis showed reduced TLT-1 expression. Isolated human T cells treated with purified soluble TLT-1 induced Activation-Induced-Cell Death (AICD) in CD8 T cells. These findings were supported by alterations in transcriptome profiling and markers including p-CD28, Bax, caspase-3. Ex-vivo TLT-1 treatment significantly (p<0.01) limited the MHC-I dependent stimulation of human CD8 T cells by a standard mix of MHC-I specific peptides. Computational modeling for molecular docking identified structural binding sites for TLT-1 on the T cell CD3 epsilon receptor. In the mouse model of ITP, injected TLT-1 reduced the circulating CD8 T cell activation (p <0.02) and protected platelets from T cell mediated destruction in vivo, while inhibition by TLT-1 blocking antibody reversed this effect (p <0.01).
Conclusion: Our results show that TLT-1 limits MHC-I mediated CD8 T cell activation and autoimmune response in ITP. Platelet TLT-1-CD3e axis can act as a novel regulatory mechanism in ITP. Soluble TLT-1 could thus be developed as a new therapeutic agent against T cell mediated platelet dysfunction in ITP and potentially in other autoimmune disorders as well, highlighting broader significance beyond ITP.